Application of urine-derived stem cells in ED treatment

Abstract

The invention relates to the field of biological medicine, and discloses application of urine-derived stem cells in ED treatment. According to the scheme, a 9-copy hypoxia response element, a minimum promoter, a vascular endothelial growth factor and a nerve growth factor gene are introduced into urine-derived stem cells by utilizing a lentiviral vector, and a hypoxia response type gene modified cell system is constructed. Through local injection of the cavernous body, the cells accurately start factor expression in an anoxic microenvironment. According to the invention, deep coupling of vascular regeneration and neural restoration can be realized, expression can be automatically reduced along with rising of tissue oxygen content, excessive hyperplasia complications can be avoided, and closed-loop intelligent treatment of pathological induction-precise intervention can be realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of urinary stem cells in the treatment of erectile dysfunction (ED). Background Technology

[0002] In the existing technological system, modifying urinary stem cells using genetic engineering to overexpress vascular endothelial growth factor (VEGF) or nerve growth factor (NGF) is currently the mainstream strategy for improving the treatment of erectile dysfunction (ED). These growth factors can improve the hemodynamic parameters of the corpora cavernosa by promoting angiogenesis and nerve fiber repair. Introducing target genes into stem cells via viral vectors or plasmids, causing them to continuously release bioactive molecules in the damaged area, is considered a solution to overcome insufficient endogenous repair capacity.

[0003] Existing gene expression systems often employ strong promoters (such as cytomegalovirus promoters) to drive the transcription of target genes, resulting in a non-specific and persistently high level of therapeutic factor expression in the host. While this high-concentration exposure may promote tissue regeneration in the early stages of treatment, prolonged and excessive vascular endothelial growth factor expression can easily lead to serious secondary problems as the repair process progresses, such as excessive local angiogenesis, structural malformations, and even tumor-like changes like hemangiomas.

[0004] Uncontrolled and persistent expression of nerve growth factor can also lead to sensitization of the sensory nervous system, resulting in unwanted pathological reactions. This uncontrolled expression, caused by the lack of feedback regulation mechanisms, makes it difficult for existing technologies to avoid dose-dependent side effects while pursuing therapeutic gains, thus limiting their safety evaluation in clinical applications.

[0005] In pathological conditions, damaged tissues are often accompanied by severe ischemia and hypoxia. When conventional stem cells enter such hypoxic areas, they often exhibit increased apoptosis rates and decreased paracrine activity due to a lack of adaptive regulatory mechanisms, leading to a shortened therapeutic window. Furthermore, because therapeutic factors generally have short biological half-lives, if precise and on-demand delivery cannot be achieved based on the degree of local hypoxia, it is difficult to maintain therapeutic concentrations, resulting in low overall repair efficiency. Summary of the Invention

[0006] This invention provides an application of urinary stem cells in the treatment of erectile dysfunction (ED), aiming to address the technical bottlenecks of existing stem cell therapies in treating ED, such as excessive angiogenesis caused by the continuous expression of exogenous therapeutic factors, low survival rate of transplanted cells in the ischemic and hypoxic microenvironment of the penile corpora cavernosa, short half-life of therapeutic factors, and inability to release them on demand. This invention constructs a gene-modified urinary stem cell system with hypoxia-responsive characteristics, achieving precise expression of therapeutic factors and deep coupling of tissue repair function in the pathological microenvironment.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides an application of urinary stem cells in the treatment of erectile dysfunction (ED), based on the construction of a hypoxia-responsive gene-modified urinary stem cell line. The urinary stem cell line uses urinary stem cells isolated from human urine as host cells, and introduces them into an expression vector driven by a hypoxia-responsive element through genetic engineering. This expression vector carries vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) genes.

[0008] Regarding the extraction and identification of urinary stem cells involved in this invention, the urinary stem cells are derived from midstream urine of healthy subjects. The specific extraction process is as follows: After collecting urine samples, centrifuge at 400g for 10 minutes at 4 degrees Celsius, discard the supernatant, and obtain cell sediment. Wash the sediment twice with phosphate-buffered saline, and then resuspend the cells in a solution containing 10% fetal bovine serum, 1% penicillin and streptomycin, 5 ng / mL epidermal growth factor, 0.4 μg / mL hydrocortisone, 5 μg / mL insulin, 5 μg / mL transferrin, and 2 × 10⁻⁶ ppm of urealyticum. -9 The cells were cultured in a complete medium containing M triiodothyronine. Primary culture was performed in a humidified incubator at 37°C and 5% CO2 concentration. When cell confluence reached 80% to 90%, passage was performed using 0.25% trypsin-EDTA digestion solution. Flow cytometry analysis revealed that the urinary stem cells showed positive expression of surface markers CD29, CD44, CD73, and CD90, with positive rates all greater than 95%; while CD31, CD34, CD45, and HLA-DR were negatively expressed, with positive rates all less than 2%. The cells possessed multi-directional differentiation potential, differentiating into osteoblasts, adipocytes, chondrocytes, and myogenic cells.

[0009] The core of constructing the hypoxia-responsive expression vector of this invention lies in building a molecular switch capable of sensing changes in oxygen partial pressure and autonomously regulating transcription intensity. The backbone of the expression vector utilizes a third-generation lentiviral vector system, and its structure, from the 5' end to the 3' end, sequentially includes: a left long terminal repeat (5'-LTR), a packaging signal (Ψ), a response element module, a minimal promoter module, a target gene expression cassette, and a right long terminal repeat (3'-LTR).

[0010] The response element module consists of multiple copies of tandem hypoxia response elements. The monomeric core sequence of the hypoxia response element is 5'-RCGTG-3' (where R is A or G). In a preferred embodiment, the response element module consists of nine tandem 5'-CCCGTG-3' motifs, with random spacers of 6 to 10 base pairs between each motif to eliminate the influence of steric hindrance on transcription factor binding. The minimal promoter module is located downstream of the response element module and uses a cytomegalovirus minimal promoter. The enhancer sequence in the full-length cytomegalovirus promoter has been removed, retaining only the TATA box and transcription start site to ensure that the target gene is at an extremely low background expression level under normoxic conditions, while achieving high-level transcriptional activation under hypoxia induction.

[0011] The target gene expression cassette contains a vascular endothelial growth factor (VEGF-165) gene sequence and a nerve growth factor (NGF-β) gene sequence linked by an internal ribosome entry site. The VEGF-165 gene is a full-length humanized cDNA sequence encoding a secreted glycoprotein that triggers a signaling cascade by binding to receptors on the surface of endothelial cells. The NGF-β gene is a gene sequence encoding a mature peptide used to maintain the survival of cavernous neurons and induce axonal regeneration.

[0012] The preparation process of the hypoxia-responsive gene-modified urinary stem cell line described in this invention is as follows: First, the constructed lentiviral expression vector and packaging plasmid (containing Gag / Pol, Rev, and VSV-G genes) are co-transfected into 293T packaging cells. The culture supernatant containing viral particles is collected and concentrated by ultracentrifugation to obtain high-titer lentiviral particles with a titer greater than 1 × 10⁻⁶. 8 TU / mL. Subsequently, urinary stem cells in the logarithmic growth phase were infected with a multiplicity of infection (MOI) ranging from 10 to 20 under conditions containing 8 μg / mL polybendazim. Twenty-four hours after infection, the medium was replaced with complete medium, and selection was performed using the puromycin resistance gene or fluorescent protein marker carried on the vector. Stable transfected cell lines obtained through selection were cultured under normoxic conditions (approximately 21% oxygen concentration), exhibiting basal metabolic levels of vascular endothelial growth factor (VEGF) and nerve growth factor (NSF) protein secretion. When the ambient oxygen concentration decreased to a hypoxic state of 1% to 3%, intracellular hypoxia-inducible factor-1α stabilized and transfected into the nucleus, binding to the hypoxia-responsive element site on the expression vector, thereby initiating large-scale transcription and translation of VEGF and NSF.

[0013] The specific application mechanism and implementation method of urinary stem cells in ED treatment described in this invention are as follows: To address the problem of deteriorated microenvironment in the corpora cavernosa of the penis in patients with erectile dysfunction (ED), this invention utilizes the pathological adaptive properties of the genetically modified urinary stem cells. When urinary stem cells are injected locally into the damaged tissue via the corpora cavernosa, the ED lesion area (such as diabetic ED or surgically induced ED) is typically accompanied by significant microcirculatory disturbances, resulting in a lower local oxygen partial pressure than normal tissue. Under this hypoxia-triggered mechanism, the transplanted urinary stem cells not only anchor themselves in the cavernous sinuses and damaged vascular endothelial regions through their adhesion molecules, but more importantly, their onboard hypoxia response system is activated.

[0014] Highly expressed and secreted vascular endothelial growth factor (VEGF) molecules act on the vascular endothelial cells of the corpus cavernosum via the paracrine pathway. VEGF specifically binds to the VEGF R2 receptor on the surface of endothelial cells, activating downstream phospholipase C-γ and phosphatidylinositol-3-kinase pathways, promoting endothelial cell proliferation, migration, and the formation of new capillary networks, thereby improving blood perfusion in the cavernous sinusoids. Simultaneously, VEGF can induce the expression and activation of endothelial nitric oxide synthase, increasing local nitric oxide production and promoting the relaxation of corpus cavernosum smooth muscle cells through the NO-cGMP pathway, thereby increasing erectile pressure.

[0015] Simultaneously, co-expressed nerve growth factor molecules act on damaged penile dorsal nerve and cavernous nerve fibers. Nerve growth factor binds to TrkA receptors at nerve endings, activating the MAPK / ERK signaling pathway, and inducing the regeneration of damaged axons and myelin repair by inhibiting neuronal apoptosis and promoting the synthesis of cytoskeletal proteins. This synchronous repair of nerves and blood vessels fundamentally rebuilds the biological basis of erectile function.

[0016] The application method described in this invention is as follows: The hypoxia-responsive gene-modified urinary stem cells are prepared into a cell suspension. The solvent for the cell suspension is medical-grade physiological saline or phenol red-free DMEM medium, and the cell density is adjusted to 1 × 10⁶ cells / mL. 7 Up to 5×10 7 During the treatment, a micro-infusion pump was used to perform local multi-point injections at 3 to 5 points selected at the base and middle segment of the corpora cavernosa. The total injection volume for each subject was controlled at 1×10⁻⁶. 6 Up to 1×10 7 Each cell.

[0017] As a core technical principle of this invention, the feedback regulation mechanism of the system greatly enhances treatment safety. With the formation of new blood vessels and the repair of nerve function, local blood circulation in the corpora cavernosa of the penis improves, and tissue oxygen content increases accordingly. When the local oxygen partial pressure recovers to near normal physiological levels, hypoxia-inducible factor-1α in urinary stem cells undergoes hydroxylation modification and is degraded by the proteasome, losing its activation effect on hypoxia-responsive elements. At this time, the transcription of vascular endothelial growth factor and nerve growth factor genes downstream of the carrier is immediately downregulated and returns to baseline levels. This closed-loop regulation mode based on local oxygen content avoids complications such as hemangioma-like hyperplasia, local tissue fibrosis, and abnormal sensory nerve sensitization caused by continuous overexpression of therapeutic factors, achieving the intelligent treatment goal of on-demand drug delivery.

[0018] The application described in this invention also relates to enhancing the long-term survival ability of urinary stem cells in the corpus cavernosum. Since hypoxia-inducible factor-1α itself has the functions of regulating cell metabolism, promoting glycolysis, and upregulating the expression of survival-promoting genes, the activation of the hypoxia response system not only drives the expression of therapeutic factors but also simultaneously enhances the tolerance of urinary stem cells to the harsh microenvironment of erectile dysfunction (ED) lesions. The apoptosis rate of gene-modified urinary stem cells under hypoxia is lower than that of unmodified cells, thus prolonging the therapeutic window period of the cells in vivo.

[0019] To further illustrate the technical details of this invention, the key engineering parameters involved in this invention are described in detail below: In the element arrangement of the expression vector, the number of hypoxia-responsive element sequences has a decisive influence on the induction ploidy rate. Experiments have confirmed that when the copy number of hypoxia-responsive elements is less than 3, the induction ploidy rate under hypoxia is insufficient to produce the secretion of factors at therapeutic concentrations; when the copy number exceeds 12, background underexpression may occur under normoxic conditions. Therefore, this invention preferably uses a 9-copy hypoxia-responsive element sequence, coupled with a minimal promoter, which can achieve 50 to 100-fold transcriptional upregulation of the target gene under hypoxia conditions while maintaining a quiescent state under normoxic conditions.

[0020] During the in vitro expansion phase of the urinary stem cells, the concentration of growth factors added to the culture medium was precisely controlled to maintain their undifferentiated state and stemness characteristics. For example, the concentration of epidermal growth factor was maintained at 5 ng / mL, which promotes cell proliferation without inducing premature differentiation; the addition of insulin and transferrin optimized the metabolic level of the cells. This optimized culture system ensured that the urinary stem cells were in an optimal physiological activity state before lentiviral infection, thus improving gene transduction efficiency.

[0021] In the formulation of the cell suspension, to prevent mechanical damage or aggregation and blockage of cells during injection, human serum albumin at a concentration of 0.5% may be added to the suspension. The addition of albumin not only provides osmotic pressure buffering but also reduces shear force damage to the cell membrane by coating the cell surface, ensuring the survival rate of cells entering the corpus cavernosum.

[0022] Furthermore, the application scheme described in this invention demonstrates unique advantages in the treatment of diabetic erectile dysfunction (ED). Since ED caused by diabetes is often accompanied by severe neuropathy and endothelial dysfunction, and the corpus cavernosum tissue is chronically under high oxidative stress and hypoxia, conventional drug treatments are extremely ineffective. This invention, through a hypoxia-responsive element system mounted on urinary stem cells, can accurately sense severe hypoxia signals in diabetic corpus cavernosum tissue, express nerve growth factor at high levels to repair damaged autonomic nerves, and express vascular endothelial growth factor to rebuild microcirculation. Its effect on improving erectile function is superior to that of simple cell transplantation.

[0023] Regarding the implementation details of the technical solution described in this invention, the following will provide a comprehensive engineering description from multiple dimensions, including gene sequence optimization, vector construction strategy, cell modification process, pharmacological mechanism of action, and application scheme, to ensure the certainty and feasibility of the solution of this invention.

[0024] At the molecular level, the vascular endothelial growth factor gene used in this invention is preferably the human vascular endothelial growth factor 165 subtype. Vascular endothelial growth factor 165 is the most biologically active and abundant subtype among the various splice variants formed by splicing the vascular endothelial growth factor-A gene. It contains a heparin-binding domain, allowing it to be secreted in a soluble form and partially bound to the extracellular matrix, forming a local concentration gradient. This is crucial for guiding the directional growth of new blood vessels. Nerve growth factor is preferably the nerve growth factor-β subtype, which has a clear function of promoting neuronal neurite growth and survival during the repair process following cavernous nerve injury. Connecting the two genes through an internal ribosome entry site element ensures that both genes are translated onto the same mRNA, thereby achieving a constant secretion ratio of vascular and nerve repair factors and achieving a synergistic therapeutic effect.

[0025] In terms of the engineering logic of vector construction, to achieve high sensitivity to oxygen pressure, the sequence design of the response element modules follows the principles of biophysical interactions. The core sequence RCGTG of each hypoxia response element serves as the binding site for the transcription factor hypoxia-inducible factor-1α, and its flanking sequences have been optimized to use 5'-CCCGTG-3'. Studies have found that increasing cytosine content helps stabilize the local chromatin structure of the binding domain and improves binding efficiency. The introduction of the spacer sequence conforms to the periodicity of the DNA double helix, ensuring that multiple hypoxia-inducible factor-1α proteins can bind in the same phase, thereby producing a synergistic transcriptional enhancement effect on the downstream cytomegalovirus minimal promoter. The cytomegalovirus minimal promoter truncates most of the enhancer elements in the original promoter, retaining only the critical region from -53bp to +75bp. This structural feature means that the promoter exhibits almost no transcriptional activity without upstream enhancer activation, thus ensuring the zero-interference characteristic of the system in normal oxygen-containing tissues.

[0026] In the bioengineering process of urinary stem cells, the parameter settings of the lentiviral infection process are crucial for obtaining high-quality therapeutic cell lines. Environmental control during the infection process requires a precise temperature of 37±0.5 degrees Celsius and a carbon dioxide concentration of 5±0.1%. To enhance the penetration of lentivirus into urinary stem cells, a polybendazim concentration of 8 μg / mL was used. This concentration, after screening, neutralizes cell membrane charge to promote viral adsorption without producing significant cytotoxicity. The screening process employed a puromycin concentration gradient from 0.5 μg / mL to 2.0 μg / mL, and continuous pressure screening for 72 hours eliminated cells that failed to integrate gene fragments, ensuring the homogeneity of the cell population.

[0027] In the systematic description of the pharmacological effects, the cell line constructed in this invention, after entering the corpus cavernosum, exhibits three stages of action: The first stage is the environmental sensing phase: the transplanted cells migrate within the corpus cavernosum sinus space, sensing the local hypoxic environment (oxygen partial pressure typically below 10 mmHg) caused by insufficient perfusion. At this time, the activity of prolyl hydroxylase in the cytoplasm is inhibited, preventing the hydroxylation of hypoxia-inducible factor-1α protein, thus avoiding the ubiquitination degradation pathway mediated by VHL protein, and rapidly accumulating intracellularly and entering the nucleus. The second stage is the factor pumping phase: after entering the nucleus, hypoxia-inducible factor-1α binds to HIF-1β to form a heterodimer, precisely recognizing and binding to multiple copies of the hypoxia-responsive element sequence on the vector of this invention, driving the explosive expression of vascular endothelial growth factor and nerve growth factor. The secreted vascular endothelial growth factor increases the perfusion area by inducing the proliferation of corpus cavernosum sinus endothelial cells; while nerve growth factor acts paracrinely on damaged corpus cavernosum nerve endings, restoring the nerve-mediated hemodynamic response. The third stage is the homeostasis recovery period: with the improvement of local microcirculation, the oxygen partial pressure in the corpus cavernosum gradually recovers. When the oxygen partial pressure exceeds the physiological threshold (e.g., 30 mmHg), the hypoxia-inducible factor-1α degradation pathway is reactivated, the expression of exogenous factors stops, and the transplanted cells enter a low-metabolic maintenance state or gradually transform into endogenous cellular components in the corpus cavernosum, such as smooth muscle cells or endothelial cells.

[0028] In the specific drug delivery procedure, to ensure the uniformity of therapeutic effect, the preparation of the cell suspension must be carried out in a sterile laminar flow hood. The needle used for injection is an ultra-fine, non-invasive 27G to 30G needle to minimize physical damage to the tunica albuginea of ​​the corpora cavernosa. The injection route is as follows: avoiding the deep dorsal vein and dorsal artery on the dorsal side of the penis, insert the needle at a 30-degree angle into one side of the corpora cavernosa, slowly inject the drug solution, and then move to the opposite side of the corpora cavernosa for symmetrical injection. This drug delivery method ensures that the cells are evenly distributed throughout the entire cavernous sinus network.

[0029] The applications described in this invention are not limited to single cell transplantation, but also include the combined use of the genetically modified urinary stem cells with biological scaffold materials. For example, cells can be loaded into a biocompatible decellularized small intestinal submucosal scaffold or collagen hydrogel, and then implanted into the corpus cavernosum defect area. The physical support of the scaffold and the biological repair function of the cells can be used to achieve comprehensive treatment of severe fibrotic erectile dysfunction.

[0030] To maintain long-term therapeutic efficacy, this invention also involves the indirect regulation of exosome function from urinary stem cells. Under hypoxic activation, genetically modified urinary stem cells not only directly secrete vascular endothelial growth factor and nerve growth factor, but their exosomes are also rich in microRNAs that promote tissue repair. This mechanism of altering the overall cellular secretome through genetic modification allows the therapeutic effect to penetrate deeper into the microstructure of the corpus cavernosum via exosomes, a natural nanocarrier, achieving comprehensive repair from macroscopic hemodynamics to microscopic molecular signaling pathways.

[0031] Regarding biosafety control, this invention also considers the impact of vector integration sites. By employing lentiviral vectors with high integration safety and performing multiple rounds of single-clone screening, cell lines that integrate into non-coding regions or transcriptionally inactive regions of the genome are selected for subsequent amplification. This further reduces the risk of endogenous gene inactivation or proto-oncogene activation that may be caused by random transgene integration.

[0032] In actual clinical pathway simulation, the treatment plan also includes preoperative assessment of the patient's penile corpora cavernosa oxygen saturation. Non-invasive near-infrared spectroscopy is used to measure the baseline oxygen level of the corpora cavernosa in a flaccid state, serving as a reference for determining the cell injection dosage and frequency. For patients with extremely low baseline oxygen saturation, the cell injection density can be appropriately increased, utilizing the powerful responsiveness of the system of this invention to achieve rapid repair.

[0033] In describing the technical features of this invention, it is essential to emphasize the absolute quiescence of the system under normoxic conditions. Through multiple base mutation screenings of the cytomegalovirus minimal promoter core sequence, this invention has determined a specific TATA box arrangement that reduces its binding affinity to non-specific transcription factors. This improvement ensures that once the patient's erectile function recovers and local oxygen levels reach target levels, the residual expression level of exogenous therapeutic factors remains far below the threshold for producing biological effects.

[0034] This invention also relates to auxiliary metabolic support during the application of urinary stem cells. Simultaneously with the injection of the cell suspension, a small dose of antioxidant can be used to reduce oxidative damage to the transplanted cells caused by peroxides generated in the corpus cavernosum due to prolonged hypoxia. This auxiliary approach complements the gene modification system of this invention, jointly ensuring a high success rate for the treatment regimen.

[0035] In future industrial applications, the hypoxia-responsive gene-modified urinary stem cells described in this invention can be pre-prepared into standardized cryopreserved cell products. Advanced programmed freezing technology and serum-free cryopreservation solution ensure that the functional integrity of the hypoxia-responsive system and cell viability are maintained after liquid nitrogen storage and thawing. This will greatly facilitate clinical applications, enabling a standardized treatment model that is ready for immediate use.

[0036] Compared with the prior art, the beneficial effects of the present invention are: 1. By introducing a hypoxia-responsive element-pMin cytomegalovirus dual regulatory module, the defect of persistent overexpression of factors caused by traditional strong promoters is overcome. The release intensity of the therapeutic factor is positively correlated with the degree of damage to the corpus cavernosum (i.e., the degree of hypoxia), and its expression is automatically downregulated after tissue repair, thus improving the biosafety of clinical applications; 2. Urinary stem cells have the advantages of wide availability, non-invasive collection, and low immunogenicity. Combined with gene modification of the hypoxia response system, their survival rate and function in the ischemic and hypoxic areas of the corpus cavernosum in ED patients have been systematically optimized. The synergistic expression of vascular endothelial growth factor and nerve growth factor works simultaneously from the two dimensions of angiogenesis and nerve repair, solving the problem of the difficulty in curing organic ED. 3. The biological therapy system of this invention has intelligent feedback capability and can autonomously adjust the output intensity according to the real-time physiological state of the damaged tissue; 4. Urinary stem cells can be collected and reinfused from the patient's own body, avoiding ethical controversies and immune rejection. Stable transfected cell lines established through the lentiviral system exhibit good genetic stability, and the expression vector design fully considers biosafety, laying the foundation for standardized and large-scale preparation of stem cell therapy products. Detailed Implementation

[0037] This invention provides an application of urinary stem cells in the treatment of erectile dysfunction (ED). This application is based on the construction and clinical translation pathway of a gene-modified urinary stem cell system with hypoxia-responsive characteristics. This system aims to address key technical challenges in ED treatment, such as a poor local microenvironment in the corpus cavernosum, uncontrolled expression of therapeutic factors, and low survival rates of transplanted cells, through the deep integration of molecular biology techniques and cell engineering technology.

[0038] The technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples, so as to ensure that those skilled in the art can fully understand and implement the present invention.

[0039] Example 1: Urinary stem cells (positive rates of CD29, CD44, CD73, and CD90 ≥96%, positive rates of CD31, CD34, CD45, and HLA-DR ≤1.5%, with normal multi-lineage differentiation potential); Hypoxia-responsive lentiviral vector (9 copies of 5'-CCCGTG-3' motif, 8bp spacer sequence, cytomegalovirus minimal promoter, internal ribosome entry site linking vascular endothelial growth factor 165 and nerve growth factor-β, protein expression ratio 4:1). Cell suspension (medical-grade saline as solvent, cell density 3×10⁻⁶) 7(each cell / mL contains 0.5% human serum albumin). Application parameters (multiple of infection 15, polybrum concentration 8 μg / mL, 29G injection needle, 3 injection sites in the corpus cavernosum, total cell volume per injection 5 × 10⁻⁶) 6 indivual); Preparation and application steps: S1: Extraction of urinary stem cells. Collect midstream urine from healthy subjects, centrifuge at 400g for 10 minutes at 4℃, wash twice with phosphate buffer, resuspend in complete culture medium (containing 10% fetal bovine serum, 5ng / mL epidermal growth factor, etc.), primary culture at 37℃ and 5% carbon dioxide, and passage with 0.25% trypsin-EDTA at 85% confluence. S2: Lentiviral vector construction and packaging. An expression vector containing 9 copies of a hypoxia-responsive element was constructed and co-transfected with the packaging plasmid into 293T cells. The supernatant was collected, filtered at 0.45 μm, and concentrated by ultracentrifugation at 80,000 g for 2 hours at 4°C, with a titer ≥1×10⁻⁶. 8 TU / mL; S3: Cell infection and selection: lentiviral particles (multiple of infection = 15) and polybendazole were added to urinary stem cells in logarithmic growth phase. The cells were infected at 37°C and 5% carbon dioxide for 24 hours, then the complete culture medium was replaced. Stable transfected cell lines were selected with puromycin. S4: Cell suspension preparation: After screening, cells are collected by centrifugation and resuspended in physiological saline containing 0.5% human serum albumin, adjusting the density to 3×10⁻⁶. 7 cells / mL; S5: In clinical application, avoid blood vessels on the dorsal side of the penis, insert the needle at a 30-degree angle into the cavernous sinuses, inject at multiple points in 3 locations, with a total cell dose of 5×10⁻⁶ cells per injection. 6 One patient was monitored for erectile function and adverse reactions after surgery.

[0040] Example 2: Same as Example 1; Preparation and application steps: Lentiviral infection multiplicity 10, the remaining steps are the same as in Example 1.

[0041] Example 3: Same as Example 1; Preparation and application steps: Lentiviral infection multiplicity 20, the remaining steps are the same as in Example 1.

[0042] Example 4: Cell suspension density 1×10 7 Cells / mL, total cell volume in a single injection 1×10⁻⁶ 6 One, the remaining components and proportions are the same as in Example 1; Preparation and application steps: Same as in Example 1.

[0043] Example 5: Cell suspension density 5×10 7 Cells / mL, total cell volume in a single injection 1×10⁻⁶ 7One, the remaining components and proportions are the same as in Example 1; Preparation and application steps: Same as in Example 1.

[0044] Example 6: The protein expression ratio of vascular endothelial growth factor to nerve growth factor was 3:1, and the other components and proportions were the same as in Example 1; Preparation and application steps: Same as in Example 1.

[0045] Example 7: The protein expression ratio of vascular endothelial growth factor to nerve growth factor was 5:1, and the remaining components and proportions were the same as in Example 1; Preparation and application steps: Same as in Example 1.

[0046] Example 8: The hypoxia-responsive vector contains 9 copies of the 5'-ACGTG-3' motif, and the remaining components and proportions are the same as in Example 1; Preparation and application steps: Same as in Example 1.

[0047] Comparative Example 1: The lentiviral vector had no hypoxia response element module and used a full-length cytomegalovirus strong promoter to drive the continuous expression of vascular endothelial growth factor and nerve growth factor. The other components were the same as in Example 1. Preparation and application steps: Same as in Example 1, but without hypoxia response regulation function.

[0048] Comparative Example 2: The expression vector contains only the vascular endothelial growth factor 165 gene and no nerve growth factor-β gene; the other components are the same as in Example 1. Preparation and application steps: Same as in Example 1.

[0049] Test method: Functional assessment: At 1, 3, and 6 months post-treatment, the intracavernosal pressure to mean arterial pressure ratio (ICP / MAP) was measured after electrical stimulation of the cavernous nerve. Tissue repair test: Six months after treatment, the activity of endothelial nitric oxide synthase in the corpus cavernosum tissue was measured; Factor expression assay: Under hypoxic and normoxic conditions, the secretion of vascular endothelial growth factor and nerve growth factor in cells was measured, and the upregulation rate was calculated. Cell safety test: Determine the apoptosis rate of genetically modified cells under hypoxic conditions; Long-term functional testing: 24 months after treatment, assess the maintenance of erectile function; Tissue homeostasis test: Detect the ratio of smooth muscle to collagen fibers in the corpus cavernosum after treatment to assess the degree of tissue fibrosis.

[0050] The test data comparisons are shown in Table 1 and Table 2.

[0051] Table 1 Comparison of ICP / MAP ratio, endothelial nitric oxide synthase activity, hypoxia factor upregulation rate, and 12-month complication rate

[0052] Table 2 Comparison of cell hypoxia apoptosis rate, 24-month erectile function maintenance rate, and smooth muscle / collagen fiber ratio.

[0053] Examples 1 to 8 utilize hypoxia-responsive elements to achieve on-demand factor expression, with vascular endothelial growth factor and nerve growth factor synergistically repairing blood vessels and nerves, and gene modification enhancing cell survival rate under hypoxia. Comparative Example 1, lacking hypoxia-responsive elements, resulted in a high incidence of complications due to continuous factor expression; Comparative Example 2, lacking synergistic nerve growth factor, exhibited incomplete tissue repair and poor long-term effectiveness.

[0054] Multiplicity of infection 15 to 20, cell density 3 × 10⁻⁶ 7 Up to 5×10 7 When the cell count / mL and the factor expression ratio are 4:1 to 5:1, the therapeutic effect and long-term efficacy are better. Among these factors, the infection multiple affects the cell transfection efficiency, cell density determines the total amount of repair factors released, and the factor expression ratio balances the rates of vascular and nerve repair. These three factors work together to ensure the overall therapeutic performance.

[0055] Compared to Comparative Example 1, which showed sustained expression of a strong promoter, the 12-month complication rate was reduced by 100%, the 24-month erectile function maintenance rate was increased by more than 17%, and the smooth muscle / collagen fiber ratio was increased by more than 40%. Compared to Comparative Example 2, which showed single-factor expression, the ICP / MAP ratio was increased by more than 21%, the endothelial nitric oxide synthase activity was increased by more than 28%, and the cell hypoxia apoptosis rate was reduced by more than 36%, meeting the clinical needs for radical treatment of ED.

[0056] In summary, this invention achieves simultaneous improvement in the efficacy, safety, and long-term effectiveness of ED treatment by coupling hypoxia-responsive gene modification with dual-factor synergistic expression, solving the core pain points of traditional stem cell therapy. It is applicable to the clinical treatment of organic ED and has good potential for industrialization.

[0057] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of urinary stem cells in the treatment of erectile dysfunction (ED), characterized in that, The application is based on the construction of a hypoxia-responsive gene-modified urine stem cell line; The urine-derived stem cell line uses urine-derived stem cells isolated from human urine as host cells and introduces them into an expression vector driven by a hypoxia-responsive element through lentivirus-mediated genetic engineering. The expression vector is loaded with vascular endothelial growth factor gene and nerve growth factor gene. The expression vector has pathological environment sensing and feedback regulation characteristics, and can autonomously regulate the transcription intensity of the vascular endothelial growth factor gene and nerve growth factor gene according to the local oxygen partial pressure level of the corpus cavernosum. The hypoxia-responsive element-driven expression vector uses a third-generation lentiviral vector system as its backbone, and its structure from the 5' end to the 3' end includes: a left long terminal repeat sequence, a packaging signal, a response element module, a minimal promoter module, a target gene expression cassette, and a right long terminal repeat sequence. The minimum promoter module is located downstream of the response element module and uses a truncated cytomegalovirus minimum promoter. This minimum promoter retains only the TATA box and transcription start site and does not contain enhancer sequences. The engineering parameters of the response element module are set as follows: the response element module consists of 9 tandem 5'-CCCGTG-3' motifs; a random interval sequence of 6 to 10 base pairs is provided between each two adjacent 5'-CCCGTG-3' motifs; the 9 tandem 5'-CCCGTG-3' motifs are coordinated with the minimum promoter.

2. The application according to claim 1, characterized in that, Under normoxic conditions, the expression vector is in a transcriptionally silent or low-background expression state; under hypoxic pathological conditions, the expression vector is activated to achieve explosive expression of therapeutic factors, and improves local microcirculation through angiogenesis and nerve repair, and automatically downregulates expression after oxygen partial pressure recovers.

3. The application according to claim 1, characterized in that, The process for obtaining and extracting the urine-derived stem cells includes: Midstream urine samples were collected from healthy subjects, and after centrifugation, the supernatant was discarded to obtain cell sediment. The cell sediment was washed twice with phosphate buffer containing a mixture of 1% penicillin and streptomycin, and then the cells were resuspended in complete culture medium for primary culture. When the cell confluence reaches 80% to 90%, passage is performed using 0.25% trypsin-EDTA digestion solution.

4. The application according to claim 3, characterized in that, The complete culture medium comprises: basal medium containing 10% fetal bovine serum, a 1% penicillin-streptomycin mixture, 5 ng / mL epidermal growth factor, 0.4 μg / mL hydrocortisone, 5 μg / mL insulin, 5 μg / mL transferrin, and 2 × 10⁻⁶ ppm. -9 M triiodothyronine.

5. The application according to claim 3, characterized in that, The primary culture conditions are a saturated humidity environment with a temperature of 37 degrees Celsius and a carbon dioxide concentration of 5%.

6. The application according to claim 1, characterized in that, The response element module consists of multiple copies of hypoxia response elements connected in series. The single core sequence of the hypoxia response element is 5'-RCGTG-3', where R is A or G.

7. The application according to claim 1, characterized in that, The target gene expression cassette contains vascular endothelial growth factor gene sequences and nerve growth factor gene sequences linked by internal ribosome entry sites.

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